The ATP-Binding Cassette Proteins of the Deep-Branching Protozoan Parasite Trichomonas vaginalis

The ATP binding cassette (ABC) proteins are a family of membrane transporters and regulatory proteins responsible for diverse and critical cellular process in all organisms. To date, there has been no attempt to investigate this class of proteins in the infectious parasite Trichomonas vaginalis. We have utilized a combination of bioinformatics, gene sequence analysis, gene expression and confocal microscopy to investigate the ABC proteins of T. vaginalis. We demonstrate that, uniquely among eukaryotes, T. vaginalis possesses no intact full-length ABC transporters and has undergone a dramatic expansion of some ABC protein sub-families. Furthermore, we provide preliminary evidence that T. vaginalis is able to read through in-frame stop codons to express ABC transporter components from gene pairs in a head-to-tail orientation. Finally, with confocal microscopy we demonstrate the expression and endoplasmic reticulum localization of a number of T. vaginalis ABC transporters

Gene expression biomarkers of heat stress in scleractinian corals: Promises and limitations

Gene expression biomarkers (GEBs) are emerging as powerful diagnostic tools for identifying and characterizing coral stress. Their capacity to detect sublethal stress prior to the onset of signs at the organismal level that might already indicate significant damage makes them more precise and proactive compared to traditional monitoring techniques. A high number of candidate GEBs, including certain heat shock protein genes, metabolic genes, oxidative stress genes, immune response genes, ion transport genes, and structural genes have been investigated, and some genes, including hsp16, Cacna1, MnSOD, SLC26, and Nf-kB, are already showing excellent potential as reliable indicators of thermal stress in corals. In this mini-review, we synthesize the current state of knowledge of scleractinian coral GEBs and highlight gaps in our understanding that identify directions for future work. We also address the underlying sources of variation that have sometimes led to contrasting results between studies, such as differences in experimental set-up and approach, intrinsic variation in the expression profiles of different experimental organisms (such as between different colonies or their algal symbionts), diel cycles, varying thermal history, and different expression thresholds. Despite advances in our understanding there is still no universally accepted biomarker of thermal stress, the molecular response of corals to heat stress is still unclear, and biomarker research in Symbiodinium still lags behind that of the host. These gaps should be addressed in future work

Cytoglobin as a Biomarker in Cancer: Potential Perspective for Diagnosis and Management

The search for biomarkers to detect the earliest glimpse of cancer has been one of the primary objectives of cancer research initiatives. These endeavours, in spite of constant clinical challenges, are now more focused as early cancer detection provides increased opportunities for different interventions and therapies, with higher potential for improving patient survival and quality of life. With the progress of the omics technologies, proteomics and metabolomics are currently being used for identification of biomarkers. In this line, cytoglobin (Cygb), a ubiquitously found protein, has been actively reviewed for its functional role. Cytoglobin is dynamically responsive to a number of insults, namely, fibrosis, oxidative stress, and hypoxia. Recently, it has been reported that Cygb is downregulated in a number of malignancies and that an induced overexpression reduces the proliferative characteristics of cancer cells. Thus, the upregulation of cytoglobin can be indicative of a tumour suppressor ability. Nevertheless, without a comprehensive outlook of the molecular and functional role of the globin, it will be most unlikely to consider cytoglobin as a biomarker for early detection of cancer or as a therapeutic option. This review provides an overview of the proposed role of cytoglobin and explores its potential functional role as a biomarker for cancer and other diseases

The classified ABC proteins of <i>T. vaginalis</i> compared with other eukaryotes.

<p>The classified ABC proteins of <i>T. vaginalis</i> compared with other eukaryotes.</p

NBDs of 65 predicted ABC proteins of <i>T. vaginalis</i>.

<p>Multiple sequence alignment and boot-strapping was performed as described in the material and methods. The boot-strap values (percentages) are displayed on all branches. The clustering of the ABC genes from families A–G is evident. The ABCE and F families of non-transporter proteins are highlighted on a lighter background for clarity.</p

The absence of full length transporters from <i>T. vaginalis</i>.

<p><b>A, B</b> genomic context of combinations of open reading frames that could encode for an intact full-length ABC transporter. Formatting is as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001693#pntd-0001693-g002" target="_blank">Figure 2</a>, with pairs of primers used to verify the genomic organization displayed as blue and orange arrows (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001693#pntd.0001693.s002" target="_blank">Table S1</a>). <b>C</b> The two genes TVAG_415980 and TVAG_415990 are separated only by an in-frame stop codon (TGA). RT-PCR analysis of mRNA with primers (black arrows) demonstrates that transcript containing both TVAG_415980 and TVAG_415990 sequences exists (lane 5, asterisk, at the same size as the genomic DNA control). Reactions lacking the RT step (lane 3), or containing primers only (lane 2) verify the specificity of the band in lane 5. <b>D</b> Confocal microscopy of <i>T. vaginalis</i> C1 cells. The four panels represent (left to right, scale bars 10 µm) bright-field images, detection of nuclear material by propidium iodide staining, overlay of the first pair, and finally, the lack of any anti-HA reactive signal in C1 cells. <b>E</b> Confocal microscopy of <i>T. vaginalis</i> C1 transformed with a plasmid containing the genomic DNA of TVAG_415980 and TVAG_415990 with the stop codon of the latter replaced by a double haemagluttinin (HA) tag. Parasites were fixed as described in the Methods, examined with a Zeiss LSM 710 confocal microscope, and visualization of HA-tagged ABC TVAG_415980_90 followed incubation with anti-HA primary and an Alexaflour-488 secondary antibody (green).</p

ABCA proteins in <i>T. vaginalis</i> and other eukaryotes show conservation of paired consensus and degenerate NBDs.

<p>Protein sequences for ABCA transporters from <i>T. vaginalis</i> and other eukaryotes had their TMD sequences removed, and in the case of full-length proteins, the sequence was bisected into N- and C-terminal halves. The alignment and boot-strapping were as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001693#pntd-0001693-g001" target="_blank">Figure 1</a>. The N-terminal NBD cluster contains almost entirely NBDs with consensus Walker and signature motifs, whereas the C-terminal cluster is almost always degenerate for at least one of the 3 motifs. For <i>T. vaginalis</i>, there are ten pairs of ABCA genes, and in all bar one case, the two members of each pair split on sequence into Group I (consensus) Walker/Signature) or Group 2 (degenerate Walker/Signature). <i>T. vaginalis</i> sequences marked with an asterisk also have an extracellular loop of 200–500 amino acids between the 1^st and 2^nd predicted TM helix as observed in many other eukaryotic ABCA proteins.</p

Localization of ABCD transporters in <i>T. vaginalis</i>.

<p>Parasites were fixed, labelled and imaged as described in the legend to <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001693#pntd-0001693-g003" target="_blank">Figure 3</a>. The endoplasmic reticulum protein BiP in C1 (<b>A</b>) was detected with primary anti-BiP antibody, whilst the hydrogenosomal protein TOM40-3 (<b>B</b>) and two ABCD transformants (TVAG_470720; <b>C</b>; and TVAG_605460; <b>D</b>) were detected by reactivity to anti-HA antibodies. The distributions of BIP, TVAG_605460, and TVAG_470720 are all similar with a dispersed pattern of staining. In contrast, the hydrogenosome membrane protein TOM 40-3 (<b>B</b>) was detected in discrete spherical organelles, consistent with the size and shape of the hydrogenosome. <b>E</b> the ABCD transporters of eukaryotes for two distinct sub-families, one of which (grey boxed) has independent evidence to support endoplasmic reticulum localization, whereas the other sub-family are peroxisomal localized.</p

The ABCE proteins of archaea and eukaroytes.

<p>Multiple sequence alignment and boot-strapping was performed as described in the material and methods. The boot-strap values (percentages) are displayed on all branches.</p

ABC proteins in sequence genomes.

1<p>these numbers are greater than those presented in a recent analysis of ABC transporters from protozoan parasites <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001693#pntd.0001693-Sauvage1" target="_blank">[31]</a>.</p>2<p>The proportion of full length ABC transporter genes is 0% in <i>T. vaginalis</i>, 69% in humans, 62% in <i>Arabidopsis</i>, 83% in yeast, 68% in <i>Giardia</i> and 0% in <i>E. coli</i> and <i>B. subtilis</i>.</p